Our knowledge of the overall diversity of the human gut microbiome has indeed been greatly improved by large-scale metagenomic assembly efforts. As a result of integrating the new taxa in the set of reference genomes, microbiomes can then be more comprehensively analyzed because the fraction of reads from a shotgun sequencing experiment that match a catalogued microbial genome-i.e., the metagenome’s mappability-increases. This type of microbial hidden diversity is efficiently targeted by large-scale isolate sequencing and metagenomic assembly efforts that have recently uncovered many previously unexplored taxa. This analogy has, however, come under question, since the dark matter in physics is thought to be a different form of matter while in microbiology undiscovered microbes have the same molecular basis as the known ones. It was for such hard-to-profile hidden taxa that the term “microbial dark matter”, inspired by physics, was initially coined. Microbiome taxa whose DNA is at least partially sequenced in the microbiome experiment but have not been described before and are phylogenetically far from genomes deposited in public databases represent another level of uncharacterized diversity. Although virome enrichment protocols have been developed and applied, viruses remain perhaps the most neglected class of members of microbial communities. Bacteriophages are particularly prone to being under-sampled due to their short genomes and biochemical properties (e.g., having an RNA or single-stranded DNA genome) that are typically not considered by standard sample preparation protocols. However, available isolation protocols are unavoidably biased towards certain classes of microbes and are successful only for a fraction of a microbiome’s biodiversity. Cultivation is less sensitive to the microbial concentrations in the sample than sequencing-based approaches and has contributed significantly to characterizing low-abundance taxa, especially when applied in a high-throughput setting. Exactly where this threshold lies depends in part on experimental choices and specific techniques for example, the dominance of host cells and DNA in the sample (e.g., biopsies from the intestinal mucosa) makes microbial taxa harder to detect and is a common problem in metagenomics experiments. These include low-abundance but potentially crucial taxa, whose genetic material is not sampled by sequencing techniques due to being present below the level of detection. At the deepest level of hidden diversity there are those members of the community that are not captured at all by the experiment, the undetected unknowns.
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